title: Complexity and differential expression of carbohydrate epitopes associated with L-selectin recognition of high endothelial venules.
abstract: Carbohydrate ligands for lymphocyte L-selectin are expressed on high endothelial venules (HEVs) in peripheral lymph nodes and sites of chronic inflammation and mediate the recruitment of lymphocytes from the blood into these tissues. In the mouse, these ligands, collectively termed the peripheral lymph node addressin (PNAd), have been shown to contain fucose, sialic acid, and sulfate and to include several HEV glycoproteins including GlyCAM-1, CD34, and MAdCAM-1. Monoclonal antibody (MAb) MECA-79, which binds a sulfate-dependent epitope, recognizes PNAd in both mouse and man. In humans, only CD34 has been identified among the glycoprotein species that react with MECA-79. Although P-selectin is highly expressed in tonsil HEVs, it was not found to react with MECA-79 or to support L-selectin-mediated lymphocyte rolling. To further characterize human PNAd, MAbs were developed against purified PNAd immunoisolated from human tonsil. MAbs JG-1, JG-5, JG-9, and JG-10, like MECA-79, bind HEVs in human tonsil and react similarly in Western blots, and JG-9 and JG-10 also block lymphocyte rolling on purified PNAd. In addition, by competitive ELISA on purified tonsil PNAd, all MAbs were found to react with overlapping epitopes. However, JG-1, JG-5, JG-9, and JG-10 do not recognize mouse PNAd, and unlike MECA-79, they recognize determinants that are sensitive to neuraminidase. Strikingly, the epitope recognized by JG-1, although abundant in tonsil and peripheral lymph node, is absent from appendix HEVs or HEVs in some samples of chronically inflamed skin, even though these HEVs are MECA-79 reactive. Moreover, although JG-5 and JG-9 react well with tonsil, peripheral lymph node, and inflamed skin HEVs, they react only with occasional endothelial cells in appendix tissues. These findings point to significant diversity in the carbohydrate determinants expressed by HEVs and recognized by L-selectin and demonstrate their differential representation in different sites in vivo. These antibodies should be useful in probing the precise structure of human L-selectin ligands.
authors.affiliation: Protein Design Labs, Inc., Mountain View, California 94043, USA. eberg@pdl.com
authors.forename: E L
authors.initials: EL
authors.lastname: Berg
authors.forename: A T
authors.initials: AT
authors.lastname: Mullowney
authors.forename: D P
authors.initials: DP
authors.lastname: Andrew
authors.forename: J E
authors.initials: JE
authors.lastname: Goldberg
authors.forename: E C
authors.initials: EC
authors.lastname: Butcher
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000187:drug effects / Q000276:immunology / Q000494:pharmacology; D000954:Antigens, Surface / Q000276:immunology; D002241:Carbohydrates / Q000276:immunology*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D015815:Cell Adhesion Molecules; D004730:Endothelium, Vascular / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D007136:Immunoglobulins; D007150:Immunohistochemistry / Q000379:methods; D019041:L-Selectin / Q000378:metabolism* / Q000502:physiology; D008214:Lymphocytes / Q000502:physiology; D008565:Membrane Proteins; D051379:Mice; D009088:Mucoproteins; D009439:Neuraminidase / Q000494:pharmacology; D014066:Palatine Tonsil / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D014699:Venules / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D002241:Carbohydrates; D015815:Cell Adhesion Molecules; D000939:Epitopes; D007136:Immunoglobulins; C069886:L-selectin counter-receptors; C504265:MADCAM1 protein, human; D008565:Membrane Proteins; D009088:Mucoproteins; D019041:L-Selectin; D009439:Neuraminidase